Immobilization of enzymes has been described by a vast number of techniques basically aiming at reducing the cost contribution of enzymes in the overall enzymatic process; facilitating recovery of enzymes from the products; and enabling continuous operation of the process.
Immobilization techniques are in general divided according to the following:    1. Physical adsorption of enzymes to solid supports, such as silica and insoluble polymers.    2. Adsorption on ion-exchange resins.    3. Covalent binding of enzymes to a solid support material, such as epoxidated inorganic or polymeric supports.    4. Entrapment of enzymes in a growing polymer.    5. Confinement of enzymes in a membrane reactor or in semi-permeable gels.    6. Cross-linking enzyme crystals (CLECS's) or aggregates (CLEAS's).
All the aforementioned enzyme immobilization procedures are comprised of the following steps:    1. Dissolving the enzyme in an appropriate buffer system with respect to pH, temperature, type of buffer salts and ionic strength.    2. Adding the solid support into the enzyme solution and mixing for some time till enzyme molecules are immobilized on the solid support.    3. Filtering off the solid support which contains the immobilized enzyme.    4. Washing the support with an appropriate buffer to remove loosely bound enzyme molecules and then drying the solid support.
Interfacial enzymes, mostly lipases, have been immobilized following the aforementioned techniques. These offered immobilized enzyme preparations possessing low synthetic activity and/or short operational half-life time. In an attempt to increase the synthetic activity and stability of immobilized lipases and other interfacial enzymes different activation methods have been applied. These methods include:    1. Binding the surface functional groups of enzymes with hydrophobic residues such as fatty acids or polyethylene glycol.    2. Coating the surface of enzymes with surfactants, such as polyol fatty acid esters.    3. Contacting enzymes with hydrophobic supports, typically polypropylene, which have been pretreated with hydrophilic solvents, such as ethanol or iso-propanol.
None of the above mentioned methods yielded satisfactory results with respect to stabilization and cost-effectiveness of immobilized interfacial enzymes, in order to carry out enzymatic reverse conversions at industrial quantities. Also, it has been reported that most enzymes, when immobilized according to the aforementioned procedures, either lose a significant portion of their synthetic activity or they do not exhibit their full activity performance due to certain constraints imposed by the immobilization procedure, or because of the presence of certain enzyme inhibitors in the reaction medium.
Another major drawback of lipases and phospholipases is their low tolerance towards hydrophilic substrates, in particular short-chain alcohols and short-chain fatty acids (below C4). It has been observed in many research studies that short-chain alcohols and short-chain fatty acids, such as methanol and acetic acid, respectively, are responsible for detaching essential water molecules from the quaternary structure of those enzymes, leading to their denaturation and consequently loss of their catalytic activity. This drawback has prohibited the application of lipases for production of commercial quantities of fatty acids methyl esters “biodiesel” using oil triglycerides and methanol as substrates.
An additional drawback of using immobilized lipases for transesterification/esterification of a fatty acid source with a free alcohol is the accumulation of the formed glycerol and water by-products on the biocatalyst and therefore prohibiting the substrates from free access to the active site of the immobilized enzyme. Such biocatalysts generally lose their catalytic performance after a few cycles when the same batch of biocatalyst is used.
The present inventors have developed special immobilized enzyme preparations, exhibiting good stability over many production cycles, persisting activity. Examples of such enzyme preparations are disclosed, inter alia, in WO/2008/084470, WO/2008/139455 and WO2009/069116.
Conditions under which the catalytic reaction is carried out, may adversely affect the stability and efficiency of immobilized enzyme preparations. It is important to have enzyme preparations which retain stability and activity under the reaction conditions.
These and other objects of the invention will become apparent as the description proceeds.